Cultures of urothelial cells provide material for physiological studies and provide tissue matrices or matrix components for the surgical reconstruction of the genitourinary tract. Methods for culturing several types of animal and human urothelial cells have been reported. Several investigators have successfully cultured human bladder epithelial cells in serum-free media with low calcium concentrations and defined growth  factors (see, e.g., Kirk, D. et al. 1985 In vitro Cell Develop Biol 21:165; Hutton, K. A. R. et al. 1993 J Urol 150:721; Petzoldt, J. L. et al. 1994 Urol Res 22:67; and Cilento, B. G. et al. 1994 J Urol 152:665). One group of investigators plated fibroblast feeder cells with human bladder epithelial cells. Another reported that medium conditioned by fibroblasts from the bladder was able to stimulate human bladder epithelial cell proliferation. Bladder epithelial cells have been cultured on various substrates, including collagen, extracellular matrix, and agar (see, e.g., Bulbul, M. A. et al. 1986 J Urol 136:512; Vatne, V. et al. 1998 Anticancer Res 18:3979). Atala, A. et al. 1993 J Urol 150:608 have achieved cell proliferation on synthetic polymers which could be implanted into animals without untoward effects.
Normal bladder includes a multilayered epithelium in which the basal layer rests on a basal lamina and the intermediate layers of cuboidal cells are covered by a superficial layer of “umbrella” cells. Umbrella cells contain fusiform vesicles, and have membranes that are asymmetrical and are comprised of uroplakin proteins (Hicks, R. H. 1975 Biol Rev 50:214). Umbrella cells line the luminal surface of the bladder, and the tight junctions joining them provide an effective barrier to the flux of particular ions. In addition, normal bladder cells express a number of specific keratins.
There is interest in the art to provide a robust in vitro multi-layered human epithelial bladder system that maintains the properties of in vivo human epithelial bladder cells, including expression of appropriate keratins. The present invention satisfies this interest.